Insect Biochemistry and Molecular Biology 30 2000 813–819 www.elsevier.comlocateibmb
Quantification of juvenile hormone III, vitellogenin, and vitellogenin-mRNA during the oviposition cycle of the lubber
grasshopper
D.W. Borst , M.R. Eskew, S.J. Wagner, K. Shores, J. Hunter, L. Luker, J.D. Hatle,
L.B. Hecht
Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
The vitellogenic cycle of the lubber grasshopper Romalea microptera was studied by measuring levels of juvenile hormone JH III, vitellogenin, and vitellogenin-mRNA through the first oviposition cycle. JH III and vitellogenin were measured by radioim-
munoassay RIA and enzyme-linked immunosorbent assay ELISA, respectively. To measure vitellogenin-mRNA, a partial 753 bp cDNA fragment of vitellogenin was isolated from the fat body of vitellogenic animals. The sequence of this cDNA was related
to vitellogenin sequences in other insect species. Using these sequence data, an RT–PCR reverse transcriptase polymerase chain reaction assay was developed to quantify vitellogenin-mRNA levels during the oviposition cycle. Vitellogenin-mRNA levels in
the fat body tissue from virgin females were measured on specific days after eclosion and compared to hemolymph levels of JH III and vitellogenin from the same individuals. The levels of all three compounds JH III, vitellogenin, and vitellogenin-mRNA
showed similar changes throughout the oviposition cycle, being undetectable or nearly undetectable initially day 3, rising to maximum levels on days 23 and 28, and then dropped to lower or undetectable levels on the day of oviposition. The ability to
measure these characteristics will be useful for studying the effects of hormonal and nutritional manipulations on reproduction.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Romalea microptera ; Vitellogenin; Juvenile hormone; JH; Oviposition cycle; RT–PCR; Vitellogenin-mRNA
1. Introduction
Numerous studies have shown that hemolymph levels of vitellogenin are hormonally regulated, and there is a
rich literature documenting how this regulation varies among insect species Engelmann, 1983; Nijhout, 1994;
Wyatt and Davey, 1996; Belles, 1998. In Orthoptera and related orders, the hemolymph concentration of juv-
enile hormone JH appears to be the critical factor that determines the onset and continuation of vitellogenesis
Chinzei et al., 1982; Wyatt and Davey, 1996. JH directly induces vitellogenin synthesis in several species
Engelmann, 1983; Wyatt and Davey, 1996. In Locusta migratoria
, JH has been shown to have both a priming
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dwborstilstu.edu D.W. Borst.
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2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 5 3 - 9
effect Wyatt et al., 1996 and a direct stimulatory effect on the transcription of vitellogenin genes in the fat body
tissue Zhang et al., 1993; Glinka and Wyatt, 1996. In addition, JH has been shown to regulate the uptake of
vitellogenin by the ovary by interacting with membrane receptors of the follicle cells Abu-Hakima and Davey,
1977; Davey et al., 1993.
One particularly attractive model for studying the regulation of vitellogenesis is the lubber grasshopper,
Romalea microptera =
guttata . Unlike many other
insect species, adult lubbers have an extended feeding and growth phase before the onset of vitellogenesis.
Thus, the events of reproduction are separated from those of molting, a process which places its own ener-
getic and regulatory demands on an organism. In addition, the large size up to 12 g of lubber grass-
hoppers makes it possible to obtain sequential hemo- lymph samples from the same individual. These samples
814 D.W. Borst et al. Insect Biochemistry and Molecular Biology 30 2000 813–819
can be analyzed for a variety of proteins and hormones, allowing the effects of environmental changes on indi-
viduals to be studied while controlling for individual variation. Indeed, a recent paper has shown that the level
of nutrition and the timing of nutritional changes have marked effects on the reproductive output of this species
Moehrlin and Juliano, 1998. Studying the effects of nutritional and hormonal manipulations on reproductive
responses should provide considerable insight into both the regulation of reproduction and the response of these
organisms to variable environments.
As the groundwork for such studies, we have analyzed the levels of JH by radioimmunoassay, RIA, vitellog-
enin by enzyme-linked immunosorbent assay, ELISA, and vitellogenin-mRNA by quantitative reverse tran-
scriptase polymerase chain reaction, RT–PCR. The sen- sitivity of these methods allowed all three factors to be
measured in each animal in this study. Our data indicate that these factors begin to rise toward the end of the
second week of the cycle, reach a peak during the fourth week of the cycle, and drop to low levels at oviposition.
These results are consistent with previous results for other Orthoptera and provide us with a baseline by which
to evaluate the effects of other hormones as well as environmental factors on the vitellogenesis of this spec-
ies.
2. Materials and methods
